Expression, Characterization, and Cellular Localization of Knowpains, Papain-Like Cysteine Proteases of the Plasmodium knowlesi Malaria Parasite
Hydrolysis of the fluorogenic substrate Z-LR-AMC by recombinant knowpains was measured at 37°C for 30 min by monitoring the release of AMC as described in the Materials and Methods section. A. Effect of pH. Hydrolysis was measured in different pH buffers (100 mM sodium acetate, pH 4.5 to 6.0; 25 mM Bis-Tris, pH 6.5; and 25 mM Tris, pH 7.0 to 8.0) containing 10 mM DTT. Activities were normalized against the maximum activity to obtain % of the maximum activity and plotted against the pH. B and C. Effect of reducing conditions. Hydrolysis was measured in the presence of different concentrations of DTT (B) or GSH (C) in 100 mM sodium acetate pH 5.5, and activities at different concentrations were normalized with the maximum activity to obtain % of the maximum activity and plotted against the concentrations of DTT or GSH. D. Effect of inhibitors. Knowpains were incubated with DMSO as a control (0.75%) or indicated inhibitors (E64, leupeptin, and pepstatin, PMSF, and EDTA) in sodium acetate buffer for 5 min at room temperature. Z-LR-AMC was added and activity was measured as described in the Materials and Methods section. Activities were expressed as % of the control and plotted against corresponding inhibitors. Data shown are means ± standard error from at least three independent experiments in triplicate.